Flavonoids in the Poisonous Plant Oxytropis falcata | Journal of

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Flavonoids in the Poisonous Plant Oxytropis falcata Wen-Hao Chen,†,‡ Rui Wang,†,‡ and Yan-Ping Shi*,†,‡ Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu ProVince, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, People’s Republic of China, and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou UniVersity, Lanzhou 730000, People’s Republic of China ReceiVed May 19, 2010

Three new flavonoids, oxytropisoflavans A (1) and B (2) and (6aR,11aR)-3,8-dihydroxy-9,10-dimethoxypterocarpan (3), together with 30 known flavonoids (4-33), were isolated from the aerial parts and roots of Oxytropis falcata. The absolute configurations of 3 and C-3 in 1 and 2 were deduced by circular dichroism. The structure of flavonoid 2 was confirmed by single-crystal X-ray diffraction analysis and that of flavonoid 3 by total synthesis of its racemate. Oxytropisoflavan A (1) is an unprecedented chalcan-isoflavan biflavonoid, whereas oxytropisoflavan B (2) possesses a rare modified A-ring. Pterocarpan 3 has good radical-scavenging activity in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Flavonoids are a group of ubiquitous and diverse molecules produced via the phenylpropanoid pathway in higher plants, and about 2% of all the photosynthesized carbon is converted into flavonoids.1 They play a variety of roles in plants including protection against UV damage and phytopathogens (e.g., phytoalexins in legumes), acting as pigments or copigments in influencing flower color, modulating auxin distribution, and as signal molecules to symbiotic microbes.2 Flavonoids often possess antioxidant activity, and the potential health benefits of fruit, vegetables, green tea, and red wine might partly be because of this property.3-5 In addition, the isoflavonoids, which are limited primarily to the Leguminosae, exhibit estrogenic and anticancer activity6,7 and, in common with the flavonoids, are also receiving considerable attention as health-promoting “nutraceuticals”. Plants of the genera Astragalus and Oxytropis (both Leguminosae) containing the indolizidine alkaloid swainsonine are commonly known as locoweeds. Consumption of the aerial plant parts of many locoweed species causes significant economic loss to cattle, sheep, and horses.8 O. falcata Bunge, known as a toxic plant, is commonly regarded as “the king of herbal medicine” in Tibetan medicine. Alcohol extracts of O. falcata mixed with other medicinal materials (Qingpenggao) have been patented as a treatment for pain and arthritis.9 It is a common paradigm in the search for new drugs in human medicine to investigate constituents of plants that have an ethnopharmacological basis for their use. Recently, a comprehensive review described the therapeutic potential of the toxic components of poisonous plants.10 In this regard, a systematic investigation of the composition of this poisonous plant has been conducted. We recently reported the occurrence of pendulone11 and a series of 24-hydroxyoleananetype triterpenoids12 and N-benzoylindole analogues13 from O. falcata. In this study we describe the isolation and characterization of three new flavonoids, oxytropisoflavans A (1) and B (2) and (6aR,11aR)-3,8-dihydroxy-9,10-dimethoxypterocarpan (3), together with 30 known flavonoids (4-33) from the aerial parts and roots of O. falcata. The radical-scavenging activity against DPPH and cytotoxic activity against the human promyelocytic leukemic (HL60) cell line of the new compounds are also described. * To whom correspondence should be addressed. Tel: +86-931-4968208. Fax: +86-931-8277088. E-mail: [email protected]. † Lanzhou Institute of Chemical Physics. ‡ Lanzhou University.

10.1021/np100339u

Results and Discussion The EtOH extracts of the aerial parts and roots of O. falcata were suspended in H2O and then partitioned successively with petroleum ether, EtOAc, and n-BuOH, respectively. Both petroleum ether-soluble fractions were combined, because they exhibited similar constituents on TLC. The organic-soluble fractions were further purified by Si gel CC and PTLC to yield three new flavonoids, oxytropisoflavans A (1) and B (2) and (6aR,11aR)-3,8dihydroxy-9,10-dimethoxypterocarpan (3). Oxytropisoflavan A (1) was obtained as an optically active, pale brown oil, with an elemental formula of C32H32O9 determined by HRESIMS (m/z 583.1944, [M + Na]+). There was evidence of hydroxy and aromatic ring absorptions in the IR spectrum at 3363, 1510, and 1463 cm-1. The 13C NMR spectrum of 1 (Table 1) displayed signals for 32 carbons, including eight sp3 carbons, 13 quaternary sp2 carbons with eight linked to an oxygen atom, and 11 tertiary sp2 carbons. The 1 H NMR spectrum of 1 (Table 1) showed four aliphatic signals at δH 4.40 (1H, d, J ) 5.2 Hz, H-R), 4.68 (1H, m, H-β), 2.73 (1H, dd, J ) 4.4, 14.0, H-γa), and 2.61 (1H, dd, J ) 7.2, 14.0, H-γb),

 2010 American Chemical Society and American Society of Pharmacognosy Published on Web 08/04/2010

Journal of Natural Products, 2010, Vol. 73, No. 8 1399 1

Table 1. H (400 MHz) and Acetone-d6a no. 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ R β γ 1′′ 2′′ 3′′ 4′′ 5′′ 6′′ 1′′′ 2′′′ 3′′′ 4′′′ 5′′′ 6′′′ 3′-OCH3 4′-OCH3 7-OH 2′-OH β-OH 2′′-OH 4′′-OH 4′′′-OH

13

C (100 MHz) NMR Data of 1 in

δH mult (J in Hz)

δC

4.23 dd (3.2, 10.0), 3.96 t (10.0) 3.48 m 2.90 dd (11.2, 15.6), 2.78 dd (4.0, 15.6) 6.85 s

69.7 t

C-4, 9

32.6 d 30.4 t

C-2, 3, 5, 9, 10, 1′

6.33 s

6.50 d (8.8) 6.83 d (8.8) 4.40 d (5.2) 4.68 m 2.73 dd (4.4, 14.0), 2.61 dd (7.2, 14.0) 6.36 d (2.4) 6.24 dd (2.4, 8.4) 7.02 d (8.4) 7.02 d (8.4) 6.72 d (8.4) 6.72 d (8.4) 7.02 d (8.4) 3.78 s 3.81 s 9.63 s 7.94 s 5.13 s 8.44 s 8.01 s 8.07 s

132.9 d 120.1 s 155.2 s 104.1 d 154.1 s 113.5 s 121.1 s 148.4 s 136.3 s 152.0 s 103.7 d 121.7 d 47.7 d 74.4 d 41.4 t 120.4 s 155.2 s 102.9 d 156.8 s 106.9 d 130.9 d 130.5 s 130.5 d 115.0 d 155.8 s 115.0 d 130.5 d 60.1 q 55.4 q

HMBC (HfC)

C-4, 7, 9, R C-6, 7, 9, 10

C-1′, 3′, 4′ C-2′, 4′ C-5, 6, 7, 1′′, 2′′, 6′′, γ C-1′′, 1′′′ C-R, β, 2′′′, 6′′′

C-1′′, 2′′, 4′′, 5′′ C-1′′, 3′′, C-R, 2′′, 4′′ C-γ, 3′′′, 4′′′, 6′′′ C-1′′′, 2′′′, 4′′′, 5′′′ C-1′′′, 3′′′, 4′′′, 6′′′ C-γ, 2′′′, 4′′′, 5′′′ C-3′ C-4′ C-6, 7, 8 C-1′, 2′, 3′ C-R, β, γ C-1′′, 2′′, 3′′ C-3′′, 4′′, 5′′ C-3′′′, 4′′′, 5′′′

a Assignments were based on DEPT135, 1H-1H COSY, HSQC, and HMBC experiments.

characteristic of a substituted propyl group in the chalcan-β-ol structure,14 and five aliphatic signals at δH 4.23 (1H, dd, J ) 3.2, 10.0, H-2a), 3.96 (1H, t, J ) 10.0, H-2b), 3.48 (1H, m, H-3), 2.90 (1H, dd, J ) 11.2, 15.6, H-4a), and 2.78 (1H, dd, J ) 4.0, 15.6, H-4b), typical of an isoflavan nucleus.15,16 These observations and the molecular mass of 560 suggested that compound 1 could be a biflavonoid possessing a chalcan-β-ol unit connected to an isoflavan unit. From analysis of the 1H-1H COSY NMR experiment, the chalcan unit of 1 appeared to be 2′′,4′′,4′′′-trihydroxylated (propterol B, 5),17 as indicated by signals at δH 6.36 (1H, d, J ) 2.4, H-3′′), 6.24 (1H, dd, J ) 2.4, 8.4, H-5′′), 7.02 (3H, d, J ) 8.4, H-6′′, 2′′′, 6′′′), and 6.72 (2H, d, J ) 8.4, H-3′′′, 5′′′), while the isoflavan

Figure 1. Key 1H-1H COSY, HMBC, and NOE correlations of 1-3.

Figure 2. CD spectra of 1-3 and 6. moiety seemed to be pentaoxygenated, with four aromatic protons resonating at δH 6.85 (1H, s, H-5), 6.33 (1H, s, H-8), 6.50 (1H, d, J ) 8.8, H-5′), and 6.83 (1H, d, J ) 8.8, H-6′), reminiscent of (3R)-(-)-isomucronulatol (6).15,16 Further supporting evidence came from 13C NMR data, which showed a close resemblance with those of propterol B (5) and (3R)-(-)-isomucronulatol (6). Connectivities of the two substructures of compound 1 were established by analysis of the HMBC spectrum as follows: The 3J HMBC cross-peaks from H-R to C-2′′/C-6′′/C-5/C-7, as well as the 2J HMBC cross-peaks from H-R to C-1′′/C-6, indicated the connection between C-6 and C-R. This was corroborated by the observation of NOESY correlations between H-R and the aromatic protons H-5 and H-6′′. The positions of the other functional groups were assigned by 1H-1H COSY, HSQC, HMBC, and NOESY (Figure 1) experiments, which resulted in the assignment of all proton and carbon signals of 1 (Table 1). The CD data of compound 1 (Figure 2) exhibited a strong positive Cotton effect for the 1Lb transition region (260-300 nm),18 indicating the 3R absolute configuration for 1. Such an allocation is strongly supported by the same Cotton effect in the CD spectrum of (3R)-(-)-isomucronulatol (6) (Figure 2).16 It should be emphasized that although the chiral chalcan unit in compound 1 is chirally perturbed by the isoflavan unit and may contribute to the lower wavelength 1La transition region (220-260 nm),18 the diagnostic 1 Lb transition region originates mainly from the isoflavan-type chromophore. The absolute configurations of C-R and C-β remain to be defined. The EIMS of compound 1 exhibited prominent fragment peaks at m/z 450, 423, 302, 107, and 180 (base peak) originated from the fission of the R,C-1′′-, R,β-, R,C-6-, and β,γ-linkage and a characteristic retro-Diels-Alder-type cleavage of the isoflavan skeleton (Scheme 1). Consequently, the structure of oxytropisoflavan A (1) was characterized as (3R)-(-)-isomucronulatol-(6fR)propterol B.

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Scheme 1. EIMS Fragmentation Pattern of 1

Table 2. 1H and 13C NMR Data of 2 and 3a 2b no.

3c

δH mult (J in Hz)

δC

2 3 4 5 6 7

4.29 dd (4.5, 9.9), 3.68 t (9.9) 3.91 m 2.02 md 1.92 md 2.67 m, 2.15 br d (17.5)

73.2 t 29.7 d 39.5 t 36.6 t 33.5 t 199.1 s

1 2 3 4 4a 6

8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 3′-OMe 4′-OMe 2′-OH 10-OH

5.24 s

108.3 d 175.5 s 65.9 s 120.6 s 149.0 s 136.8 s 152.4 s 104.0 d 122.3 d 60.9 q 56.3 q

6a 6b 7 8 9 10 10a 11a 11b 9-OMe 10-OMe

6.47 d (8.7) 6.80 d (8.7) 3.67 s 3.75 s 8.98 s 5.59 s

no.

δH mult (J in Hz) 7.33 d (8.8) 6.56 dd (2.4, 8.8) 6.36 d (2.4) 4.27 dd (5.2, 10.8), 3.67 t (10.8) 3.55 m 6.64 s

5.48 d (6.8) 3.78 s 3.89 s

δC 132.3 d 109.8 d 159.1 s 103.1 d 156.9 s 66.0 t 41.1 d 125.0 s 106.0 d 144.8 s 140.0 s 138.4 s 143.8 s 78.4 d 112.0 s 60.6 q 59.7 q

a Assignments were based on DEPT135, 1H-1H COSY, HSQC, and HMBC experiments. b In DMSO-d6 at 300 MHz for 1H and 75 MHz for 13C. c In acetone-d6 at 400 MHz for 1H and 100 MHz for 13C. d Signals overlapped.

It is important to emphasize that compound 1 is the first example of a chalcan-isoflavan biflavonoid from natural sources and also the first report of the existence of a natural biflavonoid in the genus Oxytropis. Propterol B (5) and (3R)-(-)-isomucronulatol (6) were isolated for the first time from the genus Oxytropis. Oxytropisoflavan B (2) was obtained as optically active, colorless crystals, with an elemental formula of C17H20O6 determined by HRESIMS (m/z 321.1326, [M + H]+). An IR hydroxy peak was observed at 3333 cm-1, The absorption at 1710 cm-1 and 13C NMR peak at δC 199.1 implied the presence of an R,β-unsaturated ketone

carbonyl group. The structure of the compound was established from its 1H and 13C NMR data (Table 2), which were compared to those of (3R)-(-)-isomucronulatol (6). The assignments of protons and carbons were done via 2D NMR (1H-1H COSY, HSQC, and HMBC) and DEPT experiments. It is structurally related to 6. Ring A of 2 contained an R,β-unsaturated carbonyl carbon at C-7 (δ 199.1) and the C-8 (δ 108.3), C-9 (δ 175.5) double bond characteristic of the ring A partial structure in 11b-hydroxy-11b,1dihydromedicarpin.19 Inspection of the HMBC spectrum (Figure 1) allowed for a complete assignment of all signals in this partial

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Figure 3. ORTEP drawing of 2. structure. The HMBC experiment showed that H-4 (δ 2.02, 2H, m) was related to C-1′ (δ 120.6) and C-2 (δ 73.2) and H-5 (δ 1.92, 2H, m) to C-6 (δ 33.5), C-7 (δ 199.1), C-9 (δ 175.5), and C-10 (δ 65.9). In the NOESY experiment, H-3 (δ 3.91, 1H, m) showed correlations with HO-2′ (δ 8.98, 1H, s), HO-10 (δ 5.59, 1H, m), and H-6′ (δ 6.80, 1H, d, J ) 8.7 Hz). These correlations suggested both the location and the R-orientation of the hydroxy group at C-10. The X-ray crystallographic analysis of compound 2 (Figure 3) clearly established its structure. The CD spectrum of compound 2 (Figure 2) matched very well with that of (3R)-(-)-isomucronulatol (6) at the 1Lb transition region.16,18 The (3R,10S) absolute configuration of 2 could therefore be assigned. Thus, the structure of oxytropisoflavan B (2) was established as (3R,10S)-10-hydroxy5,10-dihydroisomucronulatol. Compound 2 is the first natural product with an isoflavan skeleton possessing an R,β-unsaturated ketone carbonyl group in ring A. A similar compound was formed with protein preparation when incubated in the presence of NADPH.20 Compound 3 was obtained as a pale brown oil and was optically active ([R]D20 -155, c 0.8, acetone). In its HRESI mass spectrum, the protonated molecular ion was observed at m/z 317.1016 [M + H]+ (calcd for C17H17O5, 317.1020), corresponding to the molecular formula C17H16O5. The 1H NMR and 1H-1H COSY spectra showed four aliphatic protons in a single spin system at δH 4.27 (1H, dd, J ) 5.2, 10.8 Hz), 3.67 (1H, dd, J ) 10.8 Hz), 3.55 (1H, m), and 5.48 (1H, d, J ) 6.8 Hz), attributed to CH2-6, H-6a, and H-11a of a pterocarpan skeleton, respectively (Table 2). The presence of a pterocarpan skeleton was supported by the 13C NMR spectrum, which showed the corresponding carbons at δC 66.0 (C-6), 41.1 (C-6a), and 78.4 (C-11a).21 In the 1H NMR spectrum further signals were observed that showed the presence of two methoxy groups (δH 3.78, 3.89), an ABX aromatic proton spin system (δH 6.36, 6.56, 7.12), and a single aromatic proton (δH 6.64). The spin systems were assigned by comparison of the NMR data with those of (6aR,11aR)-3,8-dihydroxy-9-methoxypterocarpan.22 The ABX spin system corresponds to A-ring protons with the biogenetically expected oxygenation at C-3, and this was confirmed by the HMBC experiment. In the HMBC spectrum (Figure 1), correlations of the methoxy protons (δH 3.78, 3.89) with the signals at δC 140.0 and 138.4, together with the aromatic proton (δH 6.64, H-7) with the signals at δC 41.1 (C-6a), 144.8 (C-8), and 140.0 (C-9), allowed placement of the hydroxy group at C-8 and methoxy groups at C-9 and C-10, respectively. Pterocarpans are found in nature only in a 6a,11a-cis configuration. In agreement with this are the coupling constant between H-6a and H-11a (J ) 6.8 Hz) and the comparison with the literature values (J ) 6.6 Hz for cis and 13.4 Hz for trans).23 The absolute configuration at C-6a and C-11a was assigned as R from its negative optical rotation ([R]D20 -155) and the negative Cotton effect (∆ε 47.7) at 228 nm (Figure 2).24,25 Thus, compound 3 was characterized as (6aR,11aR)-3,8-dihydroxy-9,10-dimethoxypterocarpan. Pterocarpans have been gaining considerable attention on the account of their wide range of biological activities.26 In the synthesis of pterocarpans, it is essential to use methodologies that allow varying the substituents at rings A and D. Recently, a one-step,

ZnCl2-catalyzed condensation of substituted pterocarpans has been developed by Trivedi and co-workers through the reaction of 2-alkoxy-1,4-benzoquinones with appropriately substituted 2Hchromenes.27,28 To verify the structure of 3, we attempted a total synthesis of (()-3 by formal [3+2] cycloaddition reaction of 2,3alkaloxy-1,4-benzoquinone with 2H-chromene using ZnCl2 as catalyst. The synthesis of (()-3 is depicted in Scheme 2, starting from commercially available resorcinol (I) and pyrogallol (IV). Initially, I was treated with propargyl bromide to furnish II,29 which was subjected to intramolecular cyclization using PtCl2 at 100 °C to generate 2H-chromene (III).30 On the other hand, pyrogallol (IV) was first methylated with dimethyl sulfate in acetone to give 1,2,3trimethoxylbenzene (V),31 which upon oxidation with H2O2 using K3Fe(CN)6 in HOAc gave 2,3-dimethoxy-1,4-benzoquinone (VI).32 Finally, a cycloaddition reaction between III and VI, employing ZnCl2 as catalyst, afforded (()-3 with an overall of 3.2%. The structures of the synthetic compounds were elucidated mainly on the basis of comparison of their 1H and 13C NMR spectra (Supporting Information) with reported data.29-32 On the basis of NMR, EIMS, CD, optical rotation data, and comparison with reported data, the known flavonoids were identified as (6aR,11aR)-vesticarpan (4),33 propterol B (5),17 (3R)-(-)isomucronulatol (6),15,16 mucronulatol (7),34 (3R)-7,2′,3′-trihydroxy4′-methoxyisoflavan (8),35 pendulone (9),11,36 and flavonoids 10-33 (see Supporting Information). A flavonoid such as 31 was isolated for the first time from the genus Oxytropis. The accumulation of the phytoalexins medicarpin and maackiain (32) has been partly interpreted as the resistance of chickpea (Leguminosae family) to Ascochyta rabiei (a phytopathogenic deuteromycete parasite of chickpea).37 Flavonoids except for 7, 10-12, 16-18, 20, and 32 were isolated for the first time from O. falcata. The new flavonoids (1-3) were evaluated for their growth inhibition against the HL-60 cell line by the MTT method and free radical scavenging activities using the DPPH test. However, none of them exhibited significant activity on HL-60 cells with IC50 values over 50 µM. In the DPPH test, flavonoids 1 and 2 lacked significant activity, with IC20 values more than 50 µM. Compound 3 showed potent antioxidant activity, with an IC20 value of 3.8 µM, when compared with the IC20 ) 7.6 µM of the reference, Vitamin E (VE). In conclusion, the coexistence of flavonoids 2 and 6 in O. falcata further confirmed the biotransformation indicated by the related compounds obtained from the catabolism of the phytoalexin medicarpin by the chickpea pathogenic strains.20 The presence of the same substituents in equivalent positions in 3 and pendulone (9) and the co-occurrence of these compounds in O. falcata exemplify the close biosynthetic relationship between pterocarpans and isoflavonoids. This is the first report showing the potential of pterocarpans as a class of natural antioxidant. Experimental Section General Experimental Procedures. Optical rotations were measured on a Perkin-Elmer model 341 polarimeter with a 1 dm cell. Melting points were determined on an X-4 Digital Display micro-melting point apparatus, uncorrected. UV spectra were measured on a Shimadzu UV260 spectrophotometer. IR spectra were obtained on a Nicolet NEXUS 670 FT-IR spectrometer. CD spectra was recorded on an Olis DSM 1000 spectrophotometer. NMR spectra were recorded on Varian INOVA-300 and INOVA-400 spectrometers. Chemical shifts are given on the δ (ppm) scale using TMS as internal standard. EIMS and HRESIMS were carried out on a VG ZABHS mass spectrometer and a Bruker APEX II mass spectrometer, respectively. The X-ray diffraction data were collected on a Bruker Smart Apex CCD diffractometer, and the structure was solved by direct methods using Bruker SHELXS97. DPPH was obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO). Silica gel (200-300 mesh) used for column chromatography and silica GF254 (10-40 µm) for TLC were both supplied by the Qingdao Marine Chemical Factory, Qingdao, People’s Republic of China. TLC

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Scheme 2. Synthesis of (()-3a

a Reagents and conditions: (a) propargyl bromide (1 equiv), K2CO3 (3 equiv), acetone, reflux, 22 h (60%); (b) PtCl2 (0.05 equiv), toluene, 100 °C, 15 h (20%); (c) (CH3O)2SO2 (6 equiv), K2CO3 (10 equiv), acetone, reflux, 6 h (93%); (d) K3Fe(CN)6 (0.5 equiv), H2O2 (2.2 equiv), HOAc, rt, 24 h (40%); (e) ZnCl2 (1.8 equiv), DCM, rt, 2 h (72%).

was detected at 254 nm, and spots were visualized by spraying with 5% H2SO4 in C2H5OH (v/v) followed by heating. Plant Material. Oxytropis falcata was collected from Sunan County, Gansu Province, People’s Republic of China, in June 2006, and identified by adjunct Prof. Huan-Yang Qi. A voucher specimen (No. ZY-0601) has been deposited in the State Key Laboratory of Applied Organic Chemistry, Lanzhou University, People’s Republic of China. Extraction and Isolation. Plant material (aerial parts 3.5 kg, roots 0.5 kg) was air-dried, ground, and exhaustively extracted with 95% EtOH at room temperature. Each part of the plant was processed separately; the EtOH extracts of the aerial parts and roots of O. falcata were suspended in H2O and partitioned with petroleum ether, EtOAc, and n-BuOH, successively. Both petroleum ether-soluble fractions were combined because they exhibited similar constituents on TLC [Si gel; petroleum ether-EtOAc (3:1 v/v)], to give the crude petroleum ethersoluble fraction. The petroleum ether-soluble fraction (aerial parts 65 g + roots 15 g) was subjected to Si gel column chromatography (CC) eluting with petroleum ether-EtOAc (40:1) followed by stepwise addition of EtOAc to yield six fractions [Frp1 (40:1, 10 g), Frp2 (20: 1, 15 g), Frp3 (10:1, 8 g), Frp4 (5:1, 5 g), Frp5 (2:1, 9 g), Frp6 (0:1, 17 g)]. Fraction Frp1 mainly contained fatty materials, while the last fraction (Frp6, eluted with EtOAc) was a complex mixture. Fraction Frp2 was chromatographed on silica gel with a petroleum ether-EtOAc gradient system to give four subfractions (Frp2.1-Frp2.4). Further purification of subfraction Frp2.1 through repeated cheomatography with petroleum ether-EtOAc (35:1) and petroleum ether-acetone (20: 1) as eluants over Si gel yielded 10 (1 g) and 11 (50 mg). Frp2.2 was subjected to Si gel CC with petroleum ether-EtOAc (3:1) as eluant followed by recrystallization from acetone to yield 16 (6 g). Frp2.3 and Frp2.4 were separated using the same procedure as Frp2.1 to afford 20 (50 mg), 12 (130 mg), and 21 (4 mg), respectively. The EtOAc-soluble fraction (100 g) of the aerial parts of O. falcata was subjected to Si gel CC, using a step gradient-elution technique, employing mixtures of CH2Cl2-EtOAc and MeOH as solvents, to afford five fractions (Fre1 (0:1), 23 g; Fre2 (9:1), 18 g; Fre3 (4:1), 15 g; Fre4 (2:1), 16 g; Fre5 (MeOH), 20 g) according to TLC analysis. Fre1 was rechromatographed with CHCl3-EtOAc (50:1) followed by petroleum ether-actone (30:1) to give 17 (35 mg), 14 (12 mg), 13 (23 mg), and 4 (7 mg), respectively. Fre2 was subjected to Si gel CC (CHCl3-EtOAc, 20:1) to provide 25 (41 mg) and 26 (60 mg). The compounds were eluted in the following order: 7 (9 mg), 6 (5 mg), 9 (21 mg), 3 (10 mg), and 2 (18 mg). Likewise, a similar isolation procedure adopted for the EtOAc-soluble fraction (10 g) of the roots of O. falcata afforded 19 flavonoids; the pure compounds were isolated in the following order: 8 (4 mg), 33 (2 mg), 6 (540 mg), 15 (4 mg), 19 (8 mg), 18 (4 mg), 32 (28 mg), 9 (16 mg), 14 (8 mg), 28 (7 mg), 30 (3 mg), 31 (18 mg), 3 (12 mg), 22 (4 mg), 23 (4 mg), 27 (6 mg) 2 (6 mg), 5 (1 mg), and 1 (8 mg). The n-BuOH-soluble fraction (18 g) of the aerial parts of O. falcata was subjected to Si gel CC, using a step gradient-elution technique, employing mixtures of CHCl3-MeOH as solvent, to afford five fractions (Frn1 (20:1), 5 g; Frn2 (10:1), 5 g; Frn3 (4:1), 3 g; Frn4 (2: 1), 2 g; Frn5 (MeOH), 2 g) according to TLC analysis. Fraction Frn2 was chromatographed on Si gel with a CHCl3-MeOH gradient system to give three subfractions. Repeated chromatography of subfraction 2

over Si gel (CHCl3-MeOH, 8:1) followed by recrystallization afforded 24 (9 mg) and 29 (7 mg). However, the same strategy used in the separation of the n-BuOH-soluble fraction (9 g) of the roots of O. falcata resulted in no flavonoids. Oxytropisoflavan A (1): pale brown oil; [R]20 D -28 (c 0.7, acetone); IR (neat) νmax 3363, 2922, 1510, 1463, 1096 cm-1; UV (MeOH) λmax (log ε) 212 (1.63), 282 (0.27) nm; CD (MeOH, ∆ε) λmax 286 (+11.5), 261 (-22.7), 249 (-20.8) nm; 1 H and 13 C NMR data, see Table 1; EIMS (relative abundance) m/z 552 (0.3), 450 (4), 432 (11), 423 (2), 302 (59), 240 (33), 239 (32), 180 (RDA, 100), 107 (46); HRESIMS m/z 583.1944 (calcd for C32H32O9+Na, 583.1939). Oxytropisoflavan B (2): colorless, needle-like crystal (MeOH); [R]20 D +150 (c 0.2, MeOH); IR (neat) νmax 3333, 2926, 1710, 1618, 1602, -1 1180 cm ; UV (MeOH) λmax(log ε) 199.9 (1.08), 258.5 (0.50); CD (MeOH, ∆ε) λmax 302 (+24.9), 250 (+21.9), 213 (-40.5) nm; 1H and 13 C NMR data, see Table 2; EIMS (relative abundance) m/z 320 (18), 302 (6), 292 (8), 261 (15), 193 (83), 192 (64), 180 (RDA 17), 179 (40), 167 (55), 128 (100); HRESIMS m/z 321.1326 (calcd for C17H21O6, 321.1333). (6aR,11aR)-3,8-Dihydroxy-9,10-dimethoxypterocarpan (3): pale brown oil; [R]20 D -155 (c 0.8, acetone); IR (neat) νmax 3387, 2940, 1621, 1470, 1158, 1097 cm-1; UV (MeOH) λmax (log ε) 213 (1.98), 286 (0.43) nm; CD (MeOH, ∆ε) λmax 291 (+10.4), 228 (-47.7); 1H and 13C NMR data, see Table 2; HRESIMS m/z 317.1016 (calcd for C17H17O5, 317.1020). Crystallographic Data of 2. Crystallographic data for the structure of oxytropisoflavan B (2) have been deposited with the Cambridge Crystallographic Data Center as supplementary publication number CCDC 763261. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44(0)1223 336033 or e-mail: [email protected]). DPPH Scavenging Assay. To determine the antioxidant activity of the new compounds, DPPH radicals were used. In the radical form, this molecule has an absorbance at 517 nm that disappears with acceptance of an electron from an antioxidant compound to become a stable diamagnetic molecule. The method described by Chen et al.38 was used with some modifications. For each compound, different concentrations were tested. Aliquots of 30 µL of an EtOH solution containing each new compound were added to 3 mL of a 100 µM EtOH solution of DPPH. Absorbance at 517 nm was determined after 30 min at room temperature, and the percent antiradical activity (AA) was calculated using the following formula:

AA% ) 100-{[(Abssample-Absblank) × 100]/Abscontrol} EtOH (3 mL) was used as a blank. A 100 µM DPPH EtOH solution (3 mL) was used as a negative control. The IC20 value is the concentration of test sample required to scavenging 20% DPPH free radicals. VE was used as a positive control. Assessment of HL-60 Cell Viability. The HL-60 cell viability was assessed using the MTT colorimetric assay, which is based on the reduction of MTT by the mitochondrial succinate dehydrogenase of intact cells to a purple formazan product.39 Briefly, aliquots of HL-60 cells containing 5 × 104/mL were added to each well of 96-well flatmicrotiter plates and incubated with various concentrations of com-

Journal of Natural Products, 2010, Vol. 73, No. 8 1403 pounds. Six replicate wells were used in each point in the experiments. After different exposure times, MTT solution (5 mg/mL in PBS) stored at 4 °C in the dark was added to each well and plates were incubated for 4 h at 37 °C. Extraction buffer [10% SDS-0.1 M HCl, 1:1 (v/v)] was added. After an overnight incubation at 37 °C, the optical densities at 570 nm were measured by using a Bio-Rad 550 ELISA microplate reader. Acknowledgment. This project was financially supported by the Foundation for the National Key Technology Research and Development Program of China (No. 2007BAI37B05) and National Natural Science Foundation of China (No. NSFC 20621091). Supporting Information Available: Names and structures of flavonoids 10-33; IR, HR-ESIMS, 1D/2D NMR spectra of flavonoids 1-3; 1H and 13C NMR (DEPT135) of compounds II, III, V, VI, and (()-3; and the CIF file of flavonoid 2. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Harborne, J. B. Plant FlaVonoids in Biology and Medicine II: Biochemical Cellular, And Medicinal Properties; Alan R. Liss Inc.: New York. 1988; pp 17-27. (2) Taylor, L. P.; Grotewold, E. Curr. Opin. Plant Biol. 2005, 8, 317– 323. (3) Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Trends Plant Sci. 1997, 2, 152–159. (4) Hollman, P. C.; Katan, M. B. Arch. Toxicol. 1998, 20, 237–248. (5) Khalsa, K. P. S. Nutraceuticals World 1999, 2, 52–54. (6) Adlercreutz, H.; Mazur, W. Ann. Med. 1997, 29, 95–120. (7) Dixon, R. A. Isoflavonoids: Biochemistry, Molecular Biology, and Biological Functions. In ComprehensiVe Natural Products Chemistry, Vol. 1; Sankawa, U., Ed.; Elsevier, 1999; pp 773-823. (8) James, L. F.; Panter, K. E. Locoweed Poisoning in Livestock. In Swainsonine and Related Glycosidase Inhibitors; James, L. F., Elbein, A. D., Molyneux R. J., Warren, C. D., Eds.; Iowa State University Press: Ames, IA, 1989; pp 23-38. (9) Lei, J. F.; Zhang, Y. S.; Chen, L. J.; Liu, H. L.; He, G. J. Faming Zhuanli Shenqing Gongkai Shuomingshu. CN 101181372 A 20080521, 2008. (10) James, L. F.; Panter, K. E.; Gaffield, W.; Molyneux, R. J. J. Agric. Food Chem. 2004, 52, 3211–3230. (11) Chen, W. H.; Ren, Z. Y.; Wang, R.; Shi, Y. P. Anal. Sci.: X-ray Struct. Anal. Online 2008, 24, x209–x210. (12) Chen, W. H.; Qi, H. Y.; Shi, Y. P. J. Nat. Prod. 2009, 72, 1410– 1413. (13) Chen, W. H.; Wu, Q. X.; Wang, R.; Shi, Y. P. Phytochemistry 2010, 71, 1002–1006. (14) Hashimoto, F.; Nonaka, G.-I.; Nishioka, I. Chem. Pharm. Bull. 1989, 37, 77–85.

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